Preparation of thioethers of rifamycin S and rifamycin SV

ABSTRACT

New 3-thioethers of rifamycin SV are prepared by the reaction of rifamycin S with appropriate mercaptans. These thioethers of rifamycin SV are oxidized to yield the corresponding thioethers of rifamycin S. All these compounds exhibit significant antibiotic activity.

Celmer Dec. 2, 1975 PREPARATION OF THIOETHERS OF RIFAMYCIN S ANDRIFAMYCIN SV [75] Inventor: Walter D. Celmer, New London,

Conn.

[73] Assignee: Pfizer Inc., New York, NY.

[22] Filed: Apr. 1, 1974 [21] Appi. No.: 456,721

[52] U.S. Cl 260/239.3; 424/244 [51] Int. Cl. C07D 498/02 [58] Field ofSearch 260/2393 P [56] References Cited UNITED STATES PATENTS 3,625,96012/1971 Maggi 260/2393 P Primary Examiner-Henry R. Jiles AssistantExaminer-Robert T. Bond Attorney, Agent, or FirmConnolly and Hutz [57]ABSTRACT New 3-thioethers of rifamycin SV are prepared by the reactionof rifamycin S with appropriate mercaptans. These thioethers ofrifamycin SV are oxidizedto yield the corresponding thioethers ofrifamycin S. All these compounds exhibit significant antibioticactivity.

6 Claims, No Drawings BACKGROUND OF THE INVENTION The rifamycins, agroup of closely related hydroquinone-quinone antibiotics, are describedin II Farmaco, Ed. Sci. 14, 146 (1959); Antibiotics Ann. 1959/1960, 262(1960a); Experientia 16, 412 (1960); II Farmaco, Ed. Sci. 16, 165(1961); Res. Progr. Biol. med. Chem. 1, 337 (1964); and Antibiotics 1,256-264 (1967), Pergamon Press, 1st edition.

US. Pat. No. 3,625,960and II Farmaco, Ed. Sci. 22, No. 5, 307 (1967)disclose 3-thioethers of rifamycin SV in which the sulfur atom links therifamycin nucleus to complex radicals of various nature.

SUMMARY OF THE INVENTION This invention is concerned with 3-thioethersof rifamycin having the formula selected from the group consisting of MeMe B K Mo mcoo 0 m on 0 no I and wherein R is alkyl containing from 2 tocarbon atoms, allyl, phenyl and cyclohexyl.

This invention is also concerned with a process for preparing3-thioethers of rifamycin S and rifamycin SV by a sequence of stepscomprising a. contacting rifamycin S with a mercaptan of the formulaR-SH wherein R is alkyl containing from 1 to 5 carbon atoms, allyl,phenyl and cyclohexyl;

b. oxidizing the product of step (a) comprising rifamycin SV and thethioether of rifamycin SV to fonn rifamycin S and the thioether ofrifamycin S;

c. contacting the rifamycin S and the thioether of rifamycin S from step(b) with additional mercaptan of the formula R-SH;

d. repeating process steps (b) and (c) until substantial amounts ofrifamycin S starting material have been converted to the 3-thioether ofrifamycin SV;

e. oxidizing the mixture obtained from step (d) comprising a majoramount of the 3-thioether of rifamycin SV and a minor amount ofrifamycin SV and separating the 3-thioether of rifamycin S from theresulting oxidized mixture.

DETAILED DESCRIPTION OF THE INVENTION US. Pat. No. 3,625,960 and IIFarmaco, Ed. Sci. 22, No. 5, 307 (1967) describe the preparation of anumber of 3-thioethers of rifamycin SV by the process of contactingrifamycin S with an appropriate complex mercapto-compound. The failureto obtain thioethers of rifamycin SV by reacting rifamycin S with simplemercaptans (e.g. ethyl mercaptan, benzyl mercaptan, thiophenol, etc.)was explained in II Farmaco, Ed. Sci. 22, No. 5, 307 1967) as being dueto the strong reducing power of these mercaptans on the starting quinone(rifamycin S). In these instances only rifamycin SV, the hydroquinoneform of rifamycin S, could be recovered from the reaction mixture.

The present invention is concerned with process conditions forsuccessfully accomplishing the synthesis of the simple 3-thioethers ofrifamycin SV. These thioethers can be oxidized to the correspondingthioethers of rifamycin S.

The novelty of the process resides in the repeated reworking of thereaction mixture of rifamycin S with mercapto-compound by oxidizingco-produced rifamycin SV to rifamycin S which along with rifamycin Sthioether is again contacted with mercapto-compound. Appreciableremaining amounts of co-produced rifamycin SV are again oxidized torifamycin S, and the process is repeated until all the starting materialhas been substantially converted to the desired rifamycin SV thioether.Trace amounts of remaining rifamycin SV are oxidized to rifamycin Swhich is readily separated from co-produced rifamycin S thioether bycolumn chromatography. The eluted rifamycin S thioether is reduced tothe corresponding rifamycin SV thioether.

The reaction procedure is generally applicable to a range of simplemercaptans except for minor modifications in reaction temperature.Rifamycin S is dissolved in tetrahydrofuran (2% w/v) and the solution isdiluted with stirring with an equal volume of water. A simple mercaptanis added to the solution cooled to 05C. at a level of 2-5 moles ofmercaptan per mole of rifamycin S. The reaction of benzenethiol or2-methyl-2-propanethiol with rifamycin S may be conducted at 0C. to roomtemperature; the reactions with allyl mercaptan and cyclohexyl mercaptanare preferably run at room temperature. The solution is stirred forapproximately 15 minutes. Additional mercaptan is added if unreactedrifamycin S is present as determined by thin layer chromatographyemploying silica gel and a developing system of acetone-chloroform(1:1).

In addition to the 3-thioether of rifamycin SV, a considerable amount ofrifamycin SV (approximately 50%) is obtained in the reaction mixture.The material is re-worked by extracting the reaction mixture with awater-immiscible organic solvent, preferably ethyl acetate. The solventextract is dried over anhydrous sodium sulfate and the hydroquinoneantibiotics (rifamycin SV and the 3-thioether of rifamycin SV) areoxidized to the corresponding quinone antibiotics. The rifamycinquinones (rifamycin S and rifamycin S thioether),are again contactedwith mercapto-compound as previously described. The process is repeateduntil the conversion to the 3-thioether of rifamycin SV is substantiallycomplete as demonstrated by thin layer chromatography.

There is considerable difficulty in effecting the separation of the3-thioethers of rifamycin SV from small or trace amounts of rifamycin SVby column chromatography. The problem may be obviated by oxidizing allthe material to the quinone form in which state the various componentsare readily separated by column chromatography. The ethyl acetateextract of the final reaction mixture is oxidized and chromatographed ona silica gel column using ethyl acetate-hexane (3:1) as the developingsolvent. The column cuts are followed by silica gel thin layerchromatography with a developing system of acetonechloroform (1:1). Thechromatograms are examined visually. The rifamycins are highly coloredwith various shades of orange, yellow and pink.

The column cuts containing the separated 3- thioether of rifamycin S arecombined, the solvent removed under vacuum and the residue taken up inmethanol. The thioether of rifamycin S in methanol solution is reducedto the rifamycin SV thioether, the methanol removed under vacuum,residue taken up in chloroformwater and the pH adjusted to about 9 withsodium hydroxide solution. The chloroform layer is separated, dried overanhydrous sodium sulfate and taken to dryness under vacuum. The3-thioether of rifamycin SV may be crystallized from ethylacetate-hexane.

The quinone-hydroquinone system of rifamycin S and rifamycin SV isdescribed in Antibiotics 1, 256-264 (1967), pergamon Press, 1st edition.This relationship also applies for their 3-thioethers. The quinones arereadily reduced to the hydroquinones and the hydroquinones are easilyoxidized to the quinones by means well known to those skilled in theart. The preferred oxidizing agent is activated manganese dioxide whichis prepared by azeotropic drying of manganese dioxide as described in J.Org. Chem. 34, No. 6, 1979 (1969). A slurry of activated manganesedioxide is added to a methanol or ethyl acetate solution of rifamycinhydroquinone (gram/gram of antibiotic) and stirred for about 30 minutesat room termperature. The reaction mixture is clarified by filtration orcentrifugation and the solvent removed under vacuum. The preferredreducing agent is ascorbic acid which is added to a methanol or ethylacetate solution of the rifamycin quinone (2 grams/gram of antibiotic)and stirred for about 30 minutes at room temperature.

It is to be understood that the present invention includes within itsscope a number of simple 3-thioethers of rifamycin S and rifamycin SVwhich can be readily converted to one form or the other as desired. Allof these novel compounds are useful in combatting microorganisms,especially Mycobacterium tuberculosis, Diplococcus pneumoniae,Streptococcus pyogenes and Staphylococcus aureus, including strainswhich are resistant to other known antibiotics. In addition, they areuseful as disinfectants against such microorganisms and as an aid in thepurification of mixed cultures for medical diagnostic and biologicalresearch purposes.

Table I illustrates the in vitro and in vivo activity of some of thesecompounds against an antibiotic resistant strain of Staphylococcusaureus. The in vitro tests were run by preparing tubes of nutrient brothor petri dishes with nutrient agar with gradually increasingconcentrations of the pure compound, and then inoculating the media withthe specified strain of S. aureus. The minimal inhibitory concentration(M.I.C.) indicated is the minimal concentration of the compound (inmicrograms/milliliter) at which the microorganism 4 failed to grow. Thein vivo activity was determined in experimentally infected mice.

Table 1 Activity vs Staphylococcus aureus 01A0O5 *Dose that protects 50%of mice "US. 3,625,960

The corresponding thioethers of rifamycin S exhibit a comparable rangeof in vitro and in vivo activities. The more active compounds can beadministered via the oral or parenteral routes for the treatment inanimals, including humans, of pneumococcal, streptococcal,staphylococcal, tubercular and other antibiotic-sensitive infections. Ingeneral, these antibiotics are most desirably administered in daily oraldoses of 0.5-1 gram or parenteral injections of to 500 mg., depending onthe type and severity of the infection and weight of the subject beingtreated.

The compounds of this invention may be administered alone of incombination with pharmaceuticallyacceptable carriers, and suchadministration can be carried out in both single and multiple doses.

For purposes of oral administration, tablets containing variousexcipients such as sodium citrate, calcium carbonate and dicalciumphosphate may be employed along with various disintegrants such asstarch, alginic acid and certain complex silicates together with bindingagents such as polyvinylpyrrolidone, sucrose, gelatin and gum acacia.Additionally, lubricating agents such as magnesium stearate, sodiumlauryl sulfate and talc are often useful for tableting purposes. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules; preferred materials include lactose aswell as high molecular weight polyethylene glycols. When aqueoussuspensions and/or elixirs are desired for oral administration, theessential active ingredient therein may be combined with varioussweetening or flavoring agents, coloring matter or dyes, and if desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerol and variouscombinations thereof.

For purposes of parenteral administration, solutions of theseantibiotics in sesame or peanut oil or in aqueous propylene glycol maybe employed as well as sterile aqueous solutions of the correspondingwater-soluble alkali metal or alkaline-earth metal salts. Such aqueoussolutions should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose.

The 3-thioethers of rifamycin S and rifamycin SV are slightly soluble inwater and petroleum ether, and somewhat more soluble in alkalinesolutions. They are readilysoluble in methanol, ethanol, acetone andethyl acetate.

Useful analytical determinations to characterize the rifamycinthioethers of this invention included elemental analysis, ultravioletlight and infrared absorption spectra, molecular weights by themolecula'rion in the mass spectra and NMR data for thioether isomers.

' EXAMPLE I Rifamycin. S 2:%w]v).is dissolve d in tetrahydrofuran andthe solution is diluted with an equal volume of water. Methanethiol isadded as a liquid to the solution cooled to 0-5C. at a'level of :2-5moles of mercaptan per mole of rifamycin S, and stirred for aboutminutes. Additional methanethiol is added if unreacted rifamyc in Sremains as demonstrated by silica gel thin layer chromatography withadeveloping solvent system of acetone-chloroform (1:1).The reactionmixture is then extracted withethyl acetate. To the solvent extract,dried over sodium sulfate, is added activated manganese dioxide(gram/gram of antibiotic). After stirring for about 30 minuted at roomtemperature, the reaction mixture is filtered and' the solvent removedunder vacuum. The residue is again contacted with methanethiol until theconversion to 3- th iomethylrifamycin SV is substantially complete ,asdemonstrated by thin layer chromatography. The final reaction mixture isextracted with ethyl acetate. The solvent extract containing3-thiomethylrifamycin SV and traoJ amounts of rifamycin SV is oxidizedwith activated manganese dioxide, and chromatographed on a silica gelcolumn which is developed with ethyl acetate-hexane (3:1). The columncuts are followed by the use of silica gel thin layer chromatogramsdeveloped with acetonechloroforrn (1:1), and examined visually. Thecolumn cuts containing separated 3-thiomethylrifamycin S are combined,the solvent removed under vacuum and the residue taken up in methanol.Ascorbic acid (2 grams/- gram of antibiotic) is added and stirred forabout 30 EXAMPLE III A dry solid pharmaceutical composition is preparedby blending the following materials together in the proportions byweight specified below:

3-Thiomethylrifamycin SV Sodium citrate 25 Alginic acid. l0Polyvinylpyrrolidone 10 Magnesium stearate 5 EXAMPLE lV Vials areprepared containing weighed amounts of the sterile sodium salt of3-thiomethylrifamycin SV. These vials are reconstituted for parenteraladministration to 100 or 200 mg/ml with sterile water or 5% dextrosesolution.

EXAMPLE V To 3-thioethylrifamycin SV dissolved in methanol (2% w/v) isadded a slurry of activated manganese dioxide (gram/gram of antibiotic).After stirring for about 30 minutes at room temperature, the reactionmixture is filtered and the methanol removed under minutes at roomtemperature. The methanol is removed under vacuum, the residue taken upin chloroform-water and the pH adjusted to about 9 with sodium hydroxidesolution. The chloroform layer is separated, dried over anhydrous sodiumsulfate and taken to dryness under vacuum. 3Thiomethylrifamycin SV iscrystallized from ethyl acetate-hexane, yield-80%. Ultraviolet lightabsorption maxima in 0.01 N l-lCl occur at 225, 305 and 440 mu.

EXAMPLE ll vacuum to yield 3-thioethylrifamycin S. Its ultraviolet lightabsorption maxima in 0.01 N l-lCl in methanol solution occur at 220,272, 312 (inflection) and 375 my, with E values of 375, 335, I65 andrespectively. When pelleted in KBr, characteristic absorption in theinfrared region occur at the following wavelengths in microns: 2.90,3.40, 5.75, 5.95, 6.15, 6.35, 6.80, 7.05, 7.25, 7.60, 7.75, 8.00, 8.35,8.60, 9.00, 9.40, 10.30, 10.60, 10.85, 11.20, 12.20, 12.40, 12.95, 13.25and 13.90.

EXAMPLE VI The method of Example V is repeated with3-n-thiopentylrifamycin SV to yield 3-thiopentylrifamycin S withultraviolet light absorption maxima in 0.01 N l-lCl in methanol at 220,272, 312 (inflection) and 375 mg. with E 0,, values at 350, 310, 160 and80 respectively. When pelleted in KBr, characteristic absorption in theinfrared region occur at the following wavelengths in microns: 2.90,3.40, 5.75, 5.95, 6.15, 6.35, 6.80, 7.05, 7.30, 7.60, 7.75, 8.00, 8.35,8.60, 9.00, 9.40, 10.30, 10.60, 10.85, 11.20, 12.20, 12.40, 13.00 and13.90.

EXAMPLE VII The method of Example V is repeated with3-thio-tbutylrifamycin SV to yield 3-thio-t-butylrifamycin S withultraviolet light absorption maxima in 0.01 N l-lCl in methanol at 220,275, 212 (inflection) and 390 mp. with E values at 380, 290, and 60respectively.

EXAMPLE VIII The method of Example V is repeated oxidizing theappropriate 3thioethers of rifamcyin SV to yield the following:

3-thio-isopropylrifamycin S 7 3-thio-n-butylrifamycin S-3-thio-3-methyl-l-butylrifamycin S 3-thiophenylrifamycin S3-thiocyclohexylrifamcyin S 5 3-thioallylrifamycin S What is claimed is:

l. A 3-thioether of rifamycin S and rifamycin SV having the formulaeselected from the group consisting of Mo MnH wherein R is alkylcontaining from 2 to 5 carbon atoms, allyl, phenyl and cyclohexyl.

2. A process for preparing a 3-thioether of rifamycin S and rifamycin SVas claimed in claim 1, which process comprises a. contactin'g'rifamycinS in aqueous tetrahydrofuran solution at O5C. for 15 minutes with amercaptan, 2-5 molesof mercaptan per mole of rifamycin S beiplg used, ofthe formula wherein R is alkyl containing from 1 to 5 carbon atoms,allyl, phenyl or cyclohexyl;

b. oxidizing an ethyl acetate extract of the product of step (a) bystirring for 30 minutes at room temperature with 1 part of activatedmanganese dioxide per part of said product, by weight;

0. contacting the product of step (b) with additional mercaptan of theformula R-SH by the process of step (a);

i (d). repeating process steps (b) and (c) until substantial amounts ofrifamycin S starting material have been converted to a 3-thioether ofrifamycin SV;

(e) oxidizing the mixture obtained from step (d) by the process of step(b) and separating a 3-thioether of rifamycin S and of rifamycin SV byabsorption on silica gel column developed with ethyl acetatehexane(3:1).

3. 3-Thioethylrifamycin SV.

4. 3-Thio-n-propylrifamycin SV.

5. 3-Thio-isopropylrifamycin SV.

6. 3-Thio-nbutylrifamycin SV.

1. A 3-THIOETHER OF RIFAMYCIN S AND RIFAMYCIN SV HAVING THE FORMULAESELECTED FROM THE GROUP CONSISTING OF
 2. A process for preparing a3-thioether of rifamycin S and rifamycin SV as claimed in claim 1, whichprocess comprises a. contacting rifamycin S in aqueous tetrahydrofuransolution at 0*-5*C. for 15 minUtes with a mercaptan, 2-5 moles ofmercaptan per mole of rifamycin S being used, of the formula R-SHwherein R is alkyl containing from 1 to 5 carbon atoms, allyl, phenyl orcyclohexyl; b. oxidizing an ethyl acetate extract of the product of step(a) by stirring for 30 minutes at room temperature with 1 part ofactivated manganese dioxide per part of said product, by weight; c.contacting the product of step (b) with additional mercaptan of theformula R-SH by the process of step (a); (d). repeating process steps(b) and (c) until substantial amounts of rifamycin S starting materialhave been converted to a 3-thioether of rifamycin SV; (e) oxidizing themixture obtained from step (d) by the process of step (b) and separatinga 3-thioether of rifamycin S and of rifamycin SV by absorption on silicagel column developed with ethyl acetate-hexane (3:1). 3.3-Thioethylrifamycin SV.
 4. 3-Thio-n-propylrifamycin SV. 5.3-Thio-isopropylrifamycin SV.
 6. 3-Thio-n-butylrifamycin SV.